1. Field of the Invention
This invention relates to polioviruses. In particular, this invention relates to poliovirus specific primers for detection of polioviruses in clinical samples.
2. Background Art
A worldwide endeavor sponsored by the World Health Organization is underway to eradicate all wild polioviruses by the year 2000, and virologic surveillance is therefore critical to this eradication goal. In 1990, an estimated 150,000 cases of poliomyelitis were occurring annually in 70 countries where the disease is still endemic. One of the primary goals to the global eradication of poliomyelitis by the year 2000 is in the intensive surveillance of acute flaccid paralysis (AFP) which can be caused by poliovirus. This is especially true in the Americas where the spread of the wild poliovirus has ceased for a period of at least two years. Nevertheless, 2400 cases of AFP in the first 40 weeks of 1992 needed to be screened for poliovirus. Of the 60 poliovirus related cases (3% of the total), none were the wild-type virus. Twenty percent (20%) of the total cases were found to be other non-polio enteroviruses (NPEV) and the remaining cases (76%) were negative for enteroviruses. Since the surveillance of wild-type poliovirus in AFP cases must be maintained at high levels, a detection system that would identify all polioviruses rapidly to the exclusion of NPEV is needed.
NPEVs also cause a wide range of diseases in addition to AFP and the ability to distinguish these cases from vaccine-related poliovirus cases would also be very beneficial. Currently, differentiation of poliovirus from nonpoliovirus is done by limited neutralization using three types of poliovirus antisera. This procedure is time consuming and sometimes has difficulties in identifying isolates containing mixtures of poliovirus and nonpoliovirus.
Poliovirus genomes evolve rapidly during replication in humans (Nottay et al., 1981; Minor et al., 1982). As a result, the nucleotide sequences of wild polioviruses currently in circulation throughout the world are extremely heterogeneous Russ-Hess et al., 1987; Kew et al., 1990a). A typical rate for the fixation of mutations over the entire genome is one to two nucleotide substitutions per week (Nottay, et al., 1981). Although there may be a high degree of conservation at the amino acid level, there is considerable nucleotide variation. This variability occurs primarily by mutation to synonymous codons (Parvin et al., 1986), while immune selection pressures are responsible for some of this variability (Diamond et al., 1985; Blondel et al., 1986; Weigers and Dernick, 1992).
Independent wild poliovirus genotypes are usually geographically restricted (Kew et al., 1990a) and as a result, periodic epidemics involve the clonal expansion of this one restricted lineage. PCR primer sets for several wild poliovirus genotypes from the American regions have been previously described (Pan American Health Organization, 1990; Kew et al., 1990b; de Quadros et al., 1991; Yang et al., 1992). Similarly, primers have been developed which identify vaccine and reference strains of poliovirus (Yang et al., 1991; and Balanant et al., 1991). However, the molecular reagents currently in use do not allow for the rapid detection of all wild poliovirus genotypes in a single assay. Most of the PCR assays previously developed to detect either picornaviruses in general (Hyypia et al., 1989; Chapman et al., 1990; Olive et al., 1990), or polioviruses specifically (Abraham et al., 1993) have targeted conserved sequences within the 5' noncoding region. PCR primers that are specific for the 5' noncoding region are subject to possible intertypic recombination, and therefore are not applicable to world-wide detection of polioviruses due to potential crossover problems. A large proportion of vaccine-related clinical isolates are intertypic recombinants (Kew and Nottay, 1984; Minor et al., 1986a).
Until genotype-specific primers and probes can be developed for all endemic wild polioviruses, a single specific assay system is needed that 1) detects wild poliovirus genotypes, from all geographic regions, including possibly undetermined geographic regions, and 2) distinguishes NPEV infections from poliovirus infections. The ability to differentiate between poliovirus and NPEV infections is of particular importance in those regions (such as the Americas) that no longer have wild poliovirus infections, but continue to have paralytic cases due to NPEVs.
Accordingly, the present invention provides a degenerate PCR primer designed to identify all three poliovirus serotypes, while not recognizing NPEVs. The primers of the present invention are specific for polioviruses, therefore excluding all other known viruses from detection. In addition to being specific for polioviruses, the primers of the present invention are capable of detecting all poliovirus strains so far tested in all three known serotypes.
The poliovirus-specific PCR primers and methods of detection of the present invention will allow for the rapid determination of whether clinical cases of acute flaccid paralysis are the result of a polio virus infection. Therefore, this invention meets an immediate need in the worldwide poliomyelitis eradication program, since these "pan-poliovirus" primers detect all genotypes of wild and vaccine related polioviruses.
Because periodic epidemics of independent poliovirus genotypes involves clonal expansion of restricted lineages, there also exists a need to effectively track the expansion of individual serotypes of poliovirus. The molecular reagents currently in use do not allow for the rapid differentiation of individual wild poliovirus serotypes in a single assay. Serotyping is presently done using serotype-specific antisera in a micro-neutralization assay, which is time-consuming and has rather a low sensitivity level compared to molecular based methods.
Therefore, a need exists serotype-specific primers and probes can be developed for the three known serotypes of poliovirus, a for a method to rapidly distinguish between the poliovirus serotypes in order to 1) improve the speed of processing large numbers of clinical samples, and 2) increase the sensitivity of detecting minority populations of poliovirus in mixed serotype cultures.
Accordingly, the present invention provides a series of PCR primers that differentiate between the three wild poliovirus serotypes. Several sequences were identified as possible PCR primer sites after searching the poliovirus VP1 amino acid alignments that are contained in the CDC (The Centers for Disease Control and Prevention) poliovirus sequence database. Additionally, empirical evidence obtained through experimentation provided data that identified the specific degenerate PCR primers designed to identify these conserved amino acid stretches. The primers of the present invention are specific for polioviruses, therefore excluding all other known viruses from detection. In addition to being specific for polioviruses, the primers of the present invention are capable of detecting all poliovirus strains so far tested in all three known serotypes.
The sero-specific poliovirus PCR primers and methods of detection of the present invention will allow for the rapid determination of whether clinical cases of acute flaccid paralysis are the result of a polio virus infection, and allow researchers to track the spread or migration of specific poliovirus serotypes. Therefore, this invention meets an immediate need in the worldwide poliomyelitis eradication program, since these "sero-specific" poliovirus primers detect and distinguish all serotypes of wild and vaccine related polioviruses.